Download Cardiac-specific overexpression of fibroblast growth factor

Cardiac-specific overexpression of fibroblast growth factor-2 (FGF2) protects
against myocardial dysfunction and infarction in a murine model of low-flow
ischemia
Stacey L. House, Craig Bolte, Ming Zhou, Thomas Doetschman, Raisa Klevitsky,
Gilbert Newman, Jo El J. Schultz
Online Text Supplement to Methods
Generation of mice:
A.
Generation of Fgf2-deficient (KO) mice: Generation of Fgf2 KO mice,
maintained on a mixed background of 50% 129 and 50% Black Swiss, has been
previously described1.
B.
Generation of mice with a cardiac-specific overexpression of FGF2 (Tg):
The hFGF2 cDNA encoding the four human FGF2 isoforms (provided by
R. Florkiewicz) was ligated to the 3’ end of the mouse -myosin heavy chain
promoter (a gift from J. Robbins). An SV40 t-intron and polyadenylation
sequence were ligated downstream of the hFGF2 cDNA. The resulting chimeric
gene was released from the parental plasmid by Sac I and Kpn I digestion and
injected into the pronuclei of FVB/N strain fertilized mouse oocytes by the
Transgenic Mouse Facility of the University of Cincinnati. Founder mice
harboring the transgene were identified by PCR and confirmed by Southern blot
analysis. Founder mice were bred to wildtype FVB/N mice to obtain germline
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transmission. Transgenic mice were bred to wildtype FVB/N mice to maintain the
transgenic lines (MHC20 and MHC25).
Isolated Work-performing Heart Preparation:
In brief, the heart was quickly removed from the thoracic cavity and placed
in a separate preparatory tissue bath of warm (37oC), oxygenated Krebs solution.
The aorta was cannulated with a 20-gauge stainless steel cannula, preserving
the aortic valve and coronary artery ostia. Retrograde perfusion with 37.7ºC
Krebs-Henseleit solution was started immediately (Langendorff mode), at a
hydrostatic pressure of 60 mmHg. A PE-50 catheter was inserted into the left
atrium through the pulmonary vein, advanced past the mitral valve into the left
ventricle and forced through the ventricular apex. After the placement of the LV
catheter, the pulmonary vein cannula was tied into the pulmonary vein opening of
the left atrium, while carefully preserving the atrium and atrial septum. Flow was
then switched from retrograde to anterograde mode (work-performing heart
preparation), thus terminating the Langendorff perfusion. Flow through the left
atrial cannula was adjusted via a micrometer to a level that maintains pressure
inside the atrium at 6-8 mmHg (venous return = 5 mL/min) and vascular
resistance was adjusted to maintain aortic pressure at 50 mmHg, resulting in a
basal cardiac minute work of 250 mL/min*mmHg. A small cut in the pulmonary
artery proximal to the outflow tract allowed for sampling of the coronary sinus
effluent with a capillary tube to determine oxygen consumption by the heart.
Coronary arterial perfusion buffer and venous effluent samples were collected
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anaerobically, and the PO2 and PCO2 values of these samples were measured
using an automated blood gas analyzer (model 248, Ciba Corning Diagnostics
Corp). Oxygen consumption (MVO2) by the perfused hearts was computed by
multiplying the coronary flow by the arteriovenous difference in oxygen content
and normalized per gram of tissue mass as follows:
MVO2 (L O2.min-1.g-1)=(PaO2-PvO2).Coronary Flow (mL/min).(C/760)
Heart Wet Weight (g)
.
1000
where PaO2 and PvO2 represent the perfusate and venous partial pressure of O2
(mmHg), respectively, and C=0.0239 (Bunsen solubility coefficient of oxygen
dissolved in perfusate at 37°C, in milliliters of O2 per atmosphere per milliliter of
perfusate). Pacing of the heart was accomplished through electrodes connected
to the aortic and pulmonary vein cannulas. Aortic pressure, left intraventricular
pressure and left atrial pressure were measured and recorded via COBE
pressure transducers and a custom-made data acquisition system along with a
Grass polygraph. Time to peak pressure (TPP) was calculated as the time from
left ventricular end-diastolic pressure to systolic pressure. ½ Relaxation time (½
RT) was calculated as the time from left ventricular systolic pressure to ½
diastolic pressure. TPP and ½ RT were normalized to contractility and relaxation,
respectively.
Vascular Bed Staining:
Vascular density levels were determined on non-ischemic Wt, Fgf2 KO,
and FGF2 Tg hearts (4 hearts per group, 10-12 weeks of age). Paraffin-
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embedded heart sections were unmasked by digestion with pepsin (DAKO
Corporation, Carpinteria, CA) for 10 minutes at 37°C and permeabilized by
incubation in 3% hydrogen peroxide for 5 minutes. Primary antibodies raised
against -smooth muscle actin (rabbit polyclonal, 1:400 dilution, Sigma, St.
Louis, MO) and vonWillebrand’s factor (mouse monoclonal, 1:100 dilution, DAKO
Corporation, Carpinteria, CA) were utilized to stain vascular smooth muscle cells
and endothelial cells, respectively. Biotinylated secondary antibodies to rabbit
and mouse IgG (1:200 dilution, Vector Laboratories, Burlingame, CA) were then
applied to heart sections. Immunostaining was visualized utilizing the Vectastain
ABC reagent kit (Vector Laboratories, Burlingame, CA) and DAB reagent kit
(Vector Laboratories, Burlingame, CA). Sections were counterstained with
hematoxylin.
To assess the level of smooth muscle-containing blood vessels (i.e.,
arteries, veins, arterioles, and venules), sixty-four fields (four fields from each of
sixteen sections) from each -smooth muscle actin-stained mouse heart were
counted at a magnification of 20X (1.1mm2 field). Quantitation of capillary levels
(von Willebrand’s factor staining) was performed by counting 80 fields (five fields
from each of sixteen sections) from each mouse heart at a magnification of 150X
(0.06mm2 field).
Determination of FGF2 Release:
Coronary effluent was collected from Wt, Fgf2 KO, and Fgf2 Tg hearts
every 2 minutes for the last ten minutes of baseline, for the first 30 minutes and
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last 15 minutes of ischemia (60 min ischemia/120 min reperfusion protocol),
during the increase in flow (from 2 mL/min to 4 mL/min), and every 2 minutes for
the first 16 minutes and last 10 minutes of reperfusion (60 min ischemia/120 min
reperfusion protocol, see Figure 1). Quantitative determination of FGF2 release
at various time points of baseline, ischemia, and reperfusion was assessed by
ELISA according to the Quantikine human FGF basic immunoassay procedure
(R&D Systems). FGF2 concentration (pg/mL) in perfusates was normalized for
coronary flow rate and heart weight (pg/min/g heart wt).
Western blots to detect level of FGF2:
Non-ischemic Wt, Fgf2 KO, and FGF2 Tg hearts (4 hearts per group, 1012 weeks of age) were homogenized in 2 mL protein extraction buffer (20mM
Tris, 2mM EDTA, 2M NaCl, 1% NP40). Extraction of FGF2 via heparin
sepharose beads was performed on 2 mg of total protein from each heart. The
NaCl concentration of the protein samples was adjusted to 0.4M by diluting
samples in 1X TE, and then 100L of a 75% heparin sepharose bead slurry
(Amersham Pharmacia Biotech, Buckinghamshire, England) was quickly added
to each sample. Following a one hour incubation at 4°C, the heparin sepharose
beads were pelleted and washed three times with buffer containing 0.6M NaCl
and 10mM Tris-HCl ph 7.4. 15L of 10X protein sample buffer was added to the
beads and boiled for 10 minutes. The entire protein sample was then loaded on
a 15% SDS-PAGE gel and transferred to nitrocellulose. Blots were incubated
with rabbit polyclonal primary antibody to FGF2 (1:1000 dilution, Santa Cruz
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Biotechnology, Santa Cruz, CA) and then HRP-conjugated anti-rabbit IgG
secondary antibodies (1:5000 dilution, Santa Cruz Biotechnology, Santa Cruz,
CA). Results were visualized using an ECL kit (Amersham Pharmacia Biotech,
Buckinghamshire, England).
Statistical Analysis:
All values are expressed as mean±SEM. Percent functional recovery, coronary
flow, vascular density, and myocardial infarct size were compared using a oneway ANOVA followed by a Student’s t-test. Differences between functional data,
myocardial oxygen consumption values, and FGF2 release at various time points
were compared using two-way ANOVA for time and treatment with repeated
measures with Fisher’s least significant difference post-hoc test. Regression
analysis was performed to compared coronary flow with +dP/dt or infarct size.
Statistical differences were considered significant when p<0.05.
REFERENCE
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Zhou M, Sutliff RL, Paul RJ, Lorenz JN, Hoying JB, Haudenschild CC, Yin
M, Coffin JD, Kong L, Kranias EG, Luo W, Boivin GP, Duffy JJ, Pawlowski
SA, Doetschman T. Fibroblast growth factor 2 control of vascular tone. Nat
Med. 1998;4:201-7.
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